Twenty million new sexually transmitted infections (STIs) occur each year in the U.S. These infections can lead to chronic pelvic pain, stigma, as well as ectopic pregnancy and infertility. One underexplored approach for the prevention of STIs in women is harnessing the protective features of the vaginal microbiome. Abundant Lactobacillus spp. in the human vagina are thought to protect against invasion by non-indigenous bacteria, including Chlamydia trachomatis (CT), Neisseria gonorrhoeae (GC) and Trichomonas vaginalis (TV). Mechanisms underlying this protection are not well understood. Vaginal Lactobacillus spp. provide broad- spectrum antimicrobial activity in part through their production of lactic acid, which creates an acidic and hostile environment to pathogens. To date, studies have relied on bacterial cultivation techniques or microscopic evaluation to describe the association between vaginal bacteria and STI risk. The goal of this project is to identify genomic features of the vaginal microbiome and host immunological biomarkers that are associated with increased protection from incident STIs. It is hypothesized that not all Lactobacillus species or strains have the same potential to reduce the risk for STIs. This study proposes to utilize archived cervicovaginal (CVL) samples from the nested case-control Longitudinal Study of the Vaginal Microbiome Prior to Incident STI (R01-AI116799, PI: Brotman) to assess the microbial profiles of 397 women observed to acquire an incident CT, GC, or TV genital infection compared to 397 age and race matched controls. This training grant will focus on conducting metagenomic analyses of CVL samples to find genomic signatures of susceptibility to, and protection from, incident STI. The host mucosal immune response will be measured through cytokine profiling. Specific aims of this proposal are to (1) conduct a metagenomic analysis of the CVL samples to determine associations of vaginal microbial functional genes and gene pathways with incident STI risk, (2) conduct genome reconstruction analyses to characterize genomic strains of Lactobacillus spp. from vaginal metagenomes and determine their associations with STI case status, and (3) assess the interactions between human pro-inflammatory responses and the vaginal microbiome in risk for incident STI. Strain diversity may be an important modulator of the host immune response as well as host susceptibility to infection. Preliminary data for Lactobacillus iners strains show major genomic diversity between women, but strain and health outcome relationships remains to be determined. The proposed high-resolution metagenomic analyses will produce unprecedented insights into how the vaginal microbiome confers protection against acquisition of STIs and the roles of the associated host immune response. Host and microbial targets identified through these aims can be exploited in the future for the development of translational curative or prophylactic interventions that would directly affect women?s reproductive health. This fellowship will provide the applicant new training in comparative genomics and molecular epidemiology.